Von Frey Hair Test Research Articles

Glial activation in pain and emotional processing regions in the nitroglycerin mouse model of chronic migraine.

Our aim was to survey astrocyte and microglial activation across four brain regions in a mouse model of chronic migraine. Chronic migraine is a leading cause of disability, with higher rates in females. The role of central nervous system neurons and glia in migraine pathophysiology is not fully elucidated. Preclinical studies have shown abnormal glial activation in the trigeminal nucleus caudalis of male rodents. No current reports have investigated glial activation in both sexes in other important brain regions involved with the nociceptive and emotional processing of pain. The mouse nitroglycerin model of migraine was used, and nitroglycerin (10 mg/kg) or vehicle was administered every other day for 9 days. Prior to injections on days 1, 5, and 9, cephalic allodynia was determined by periorbital von Frey hair testing. Immunofluorescent staining of astrocyte marker, glial fibrillary protein (GFAP), and microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), in male and female trigeminal nucleus caudalis, periaqueductal gray, somatosensory cortex, and nucleus accumbens was completed. Behavioral testing demonstrated increased cephalic allodynia in nitroglycerin- versus vehicle-treated mice. An increase in the percent area covered by GFAP+ cells in the trigeminal nucleus caudalis and nucleus accumbens, but not the periaqueductal gray or somatosensory cortex, was observed in response to nitroglycerin. No significant differences were observed for Iba1 staining across brain regions. We did not detect significant sex differences in GFAP or Iba1 quantification. Immunohistochemical analysis suggests that, at the time point tested, immunoreactivity of GFAP+ astrocytes, but not Iba1+ microglia, changes in response to chronic migraine-associated pain. Additionally, there do not appear to be significant differences between males and females in GFAP+ or Iba1+ cells across the four brain regions analyzed.

OA29 Epigenetic regulation of IL-4 promoter targeting p300 attenuating intervertebral disc degeneration and pain by inhibiting IL-1β

Abstract Background/Aims Low back pain (LBP) is a prevalent condition primarily caused by intervertebral disc degeneration (IVDD), which imposes a substantial social and medical burden. Effective targeted treatments to prevent the progression of IVDD and alleviate LBP are still lacking. p300, a critical member of the histone acetyltransferase (HAT) family, is involved in the regulation of inflammation. This study aims to investigate the role of p300/IL-4 in IVDD and pain and elucidate the underlying molecular mechanism. Methods Human intervertebral disc samples were collected and graded based on the Pfirrmann grading scheme. A rat model of IVDD was established through posterior disc puncture, and various treatments were administered. Human nucleus pulposus cells (NPCs) were isolated and cultured, followed by assessments including qRT-PCR, Western blotting, immunofluorescence, and histopathological analyses. The mechanical and thermal hyperalgesia were assessed using von Frey hair tests and the plantar test, respectively. Chromatin immunoprecipitation (ChIP) and ChIP-qPCR were performed, and lentivirus transduction was used for p300 knockdown. Results We investigated the role of CREB binding protein (CBP)/p300 in IVDD. Degenerated nucleus pulposus (NP) tissues from IVDD patients exhibited a significant downregulation of p300 at both nucleic acid and protein levels, while CBP levels remained unchanged. Manipulating p300 expression in NPCs regulated matrix production, with p300 overexpression enhancing aggrecan and collagen II expression and knockdown leading to increased matrix-degrading proteins. In a rat IVDD model, activating p300 function with CTPB (p300-specific activator) alleviated IVDD and pain, while inhibiting p300 with C646 exacerbated IVDD and pain. Transcriptome analysis implicated IL-4 as a downstream effector of p300, and IL-4 demonstrated protective effects against IVDD and pain. Mechanistically, p300 was found to acetylate H3K27 at the IL-4 promoter, promoting its expression. Conclusion The novel findings of this study suggest that the p300/IL-4/IL-1β axis may represent a promising therapeutic target for IVDD and LBP. These results propose a hypothesis that, early in the inflammatory phase, NPCs may initiate a self-protective mechanism through p300, aiding itself in preserving a favourable metabolic microenvironment. This mechanism is primarily mediated by the expression of IL-4, which has the capacity to counteract IL-1β, rather than TNF-α. Disclosure H. Cui: None. Z. Zheng: None.

Hydroethanolic Extract of Capparis spinosa Amends Vincristine-Induced Peripheral Neuropathy in Rats

Background: Peripheral neuropathy is a common side effect of chemotherapeutic agents. This study aimed to assess the impact of hydroalcoholic extract from Capparis spinosa fruit on mechanical allodynia and heat hyperalgesia induced by vincristine. Methods: Vincristine (0.1 mg/kg/day, intraperitoneally) was used to induce peripheral neuropathy in rats for 2 weeks. The rats received treatment with the hydroalcoholic extract of Capparis spinosa fruit (100, 200, and 400 mg/kg, orally) or pregabalin (20 mg/kg, orally) for 2 weeks. Assessments of thermal and mechanical hyperalgesia were conducted using the hot plate, open field, footprint, grip strength, and von Frey hair tests. To investigate the roles of opioid and serotonergic pathways in anti-nociceptive and locomotor activities, naloxone (1 mg/kg) and cyproheptadine (5 mg/kg) were administered in conjunction with the extract. Results: Two weeks post vincristine administration, there was a significant decrease in thermal and mechanical nociceptive thresholds, muscle strength, and locomotor activity in rats compared to untreated groups. In all tests, pregabalin mitigated vincristine's effects; notably, the Capparis spinosa extract at a dose of 400 mg/kg significantly reduced neuropathic behavioral changes compared to animals that received vincristine. The efficacy of the extract at the 400 mg/kg dose was diminished by naloxone in the von Frey filament test (P < 0.01), hot plate test (P < 0.05), and grip strength test (P < 0.001). Additionally, cyproheptadine reversed the extract's effect in the grip strength (P < 0.0001) and von Frey filament tests (P < 0.01). Conclusions: The findings suggest that the hydroalcoholic extract of Capparis spinosa fruit may counteract vincristine-induced neuropathic pain through opioidergic and serotonergic pathways. The extract's beneficial effects are likely due to its flavonoid and phenolic compound content.

Small fibre integrity and axonal pathology in the rat model of experimental autoimmune neuritis.

Experimental autoimmune neuritis is a common animal model for acute human immune-mediated polyneuropathies. Although already established in 1955, a number of pathophysiological mechanisms remain unknown. In this study, we extensively characterize experimental autoimmune neuritis progression in Lewis rats, including new insights into the integrity of small nerve fibres, neuropathic pain and macrophage activation. Acute experimental autoimmune neuritis was induced with P253-78 peptide and consequently investigated using the gait analysis system CatWalk XT, electrophysiological and histopathological analyses, quantitative polymerase chain reaction (PCR), dorsal root ganglia outgrowth studies, as well as the von Frey hair and Hargreaves tests. For the longitudinal setup, rats were sacrificed at Day (d) 10 (onset), d15 (peak), d26 (recovery) and d29 (late recovery). We confirmed the classical T-cell and macrophage-driven inflammation and the primarily demyelinating nature of the experimental autoimmune neuritis. The dual role of macrophages in experimental autoimmune neuritis is implicated by the high number of remaining macrophages throughout disease progression. Furthermore, different subpopulations of macrophages based on Cx3-motif chemokine receptor 1 (Cx3cr1), platelet factor 4 (Pf4) and macrophage galactose-type lectin-1 (Mgl1) expressions were identified. In addition, modulation of the sensory system in experimental autoimmune neuritis was detected. An outgrowth of small fibres in the plantar skin at the onset and peak of the experimental autoimmune neuritis was evident parallel to the development of acute hyperalgesia mediated through transient receptor potential vanilloid 1 modulation. Our data depict experimental autoimmune neuritis as a primary demyelinating disease with implicated axonal damage, a small unmyelinated fibre impairment throughout the disease progression course, and underline the pivotal role of macrophages in the effector and during the recovery stage.

A52 SEX DIFFERENCES IN POST-INFLAMMATORY VISCERAL PAIN IN A MOUSE MODEL OF INFLAMMATORY BOWEL DISEASE (IBD)

Abstract Background Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract. Despite achieving endoscopic remission, up to 50% of IBD patients continue to experience chronic abdominal pain, with female patients displaying an increased prevalence. The reason underlying these differences in pain perception is unknown, but the influence of sex hormones represents an important biological source for variability in pain sensitivity. To date, few studies have examined sex differences in chronic visceral pain in IBD. Purpose Examine sex-specific differences in post-inflammatory chronic visceral pain in IBD. Method We used the post-inflammatory DSS mouse model of chronic visceral pain. Ovariectomy was performed to study the effects of estrogen deficiency; sham surgery was performed as a control. Male, cycling female and ovariectomized female mice were given 2.5% DSS for five days and allowed to recover for 5 weeks. Somatic pain was evaluated using the hot plate and von Frey hair tests. Visceral pain was evaluated using the visceral motor reflex (VMR) to colorectal distension five weeks after DSS treatment. Visceral and somatic pain testing in cycling females was performed in diestrus. Result(s) Male, cycling female and ovariectomized female mice given DSS initially lost weight when compared to controls (p<0.0001). Cycling females displayed significantly decreased colitis severity when compared to males [Disease Activity Index at Day 12: 1.41 ± 0.41 cycling females, n=12; 4.41 ± 0.31 males, n=12; p<0.001] but increased severity compared to ovariectomized females [Disease Activity Index at Day 12: 2.0 ± 0.41 ovariectomized females, n=13; 4.77 ± 0.6 cycling females, n=13, p=0.0005]. Increased visceral hypersensitivity was seen in post-inflammatory cycling females compared to post-inflammatory males [VMR at 60mmHg, post-inflammatory cycling females 0.10 ± 0.016, n=10; post-inflammatory males 0.07 ± 0.007, n=10; p=0.032] and post-inflammatory ovariectomized females [VMR at 60mmHg, post-inflammatory sham females 0.072 ± 0.005, n=8; post-inflammatory ovariectomized females 0.047 ± 0.005, n=8; p=0.019]. Thermal hyperalgesia and mechanical allodynia were similar across all groups. Conclusion(s) These data suggest that estrogen plays an important role in the severity of colitis severity and post-inflammatory visceral pain. Understanding sex-specific differences in post-inflammatory visceral pain in IBD may allow us to define novel therapeutic approaches for IBD patients. Disclosure of Interest None Declared

Detailed statistical analysis plan for the neurological complications in endoscopic versus open radial artery harvest (NEO) randomised clinical trial.

Coronary artery bypass grafting can be conducted using the radial artery as a bypass graft. However, it remains unclear which harvesting method is superior, i.e. endoscopic or open radial artery, and which site for proximal anastomosis of the radial artery has the greatest benefits? The NEO Trial is a single site randomised clinical trial with a 2 × 2 factorial design. The first comparison assesses endoscopic versus open radial artery harvest with a primary outcome of hand function and secondary outcomes of neurological deficits through clinical exams and neurophysiological studies. The primary outcome is postoperatively hand function at three months. We anticipate a mean difference of 3 points with a standard deviation of 8 points, a power of 90%, and a type I error of 5%, resulting in a required sample size of 300 participants randomised 1:1. Secondary outcomes are neurological deficits (based on nerve conduction measurements, algometry test and von Frey hair test), clinical neurological examination of cutaneous sensibility, and registration of complications in the donor arm (haematoma formation, wound dehiscence, and/or infection). The second comparison assesses two different proximal anastomotic sites, i.e. aorto-radial anastomosis versus mammario-radial anastomosis. The primary outcome is a composite of cerebrovascular events and the secondary outcome is graft patency evaluation by multi-slice computer tomography-scan. These outcomes will be assessed at 1year postoperatively, and the results of this comparison will be exploratory only. Both comparisons will be analysed using intention-to-treat and intervention groups will be compared using linear regression, logistic regression, or Mann-Whitney U test depending on data type. Two independent statisticians will follow the present plan and conduct the analyses which will hereafter be fused into a final analysis based on consensus. This detailed analysis plan will increase the validity of the NEO trial results by predefining the statistical analysis in detail. ClinicalTrials.gov identifier: NCT01848886 . Registered 25 February 2013. Danish Ethics committee number: H-3-2012-116. Danish Data Protection Agency: 2007-58-0015/jr. n:30-0838.

Expression and Role of CXCL13 and MiR-186-5p in the Trigeminal Ganglion of a Rat Model of Trigeminal Neuralgia.

This study sought to reveal the roles of CXCL13 and miR-186-5p in a rat model (adult male Sprague-Dawley rats, 7-8 weeks old, 180-200 g) of trigeminal neuralgia (TN) established via chronic constriction injury of the infraorbital nerve (ION-CCI). The results of behavioural tests and the expression levels of miR-186-5p and CXCL13 in the trigeminal ganglion (TG) were compared between the sham and ION-CCI groups, as well as the consequences of the miR-186-5p mimic and inhibitor. Compared with the sham-operated rats, ION-CCI rats displayed mechanical hypersensitivity in the von Frey hair test. Western blotting revealed the upregulation of CXCL13 and downregulation of miR-186-5p in the TG of ION-CCI rats relative to their expression in sham rats. Furthermore, an miR-186-5p mimic decreased CXCL13 protein levels and increased the mechanical withdrawal thresholds of ION-CCI rats. CXCL13 protein levels also increased after the injection of an miR-186-5p inhibitor. Finally, miR-186-5p was found to be expressed in the TG and was downregulated in ION-CCI rats compared to sham rats. miR-186-5p may negatively regulate CXCL13 to influence the occurrence and development of TN. Collectively, our findings shed new light on novel therapies for the treatment of TN.

Adult-onset hypothyroidism causes mechanical hypersensitivity due to peripheral nerve hyperexcitability based on voltage-gated potassium channel downregulation in male mice.

Thyroid hormones play an important role in the central and peripheral nervous system functions. Approximately 50% of adult-onset hypothyroid patients have sensory symptoms including pain, possibly caused by peripheral neuropathy. However, the mechanism causing the pain has not been clarified. We generated an adult-onset hypothyroid model animal by administering 50ppm propylthiouracil (PTU) for 5weeks to male mice. Female mice were not tested in this study. Mechanical hypersensitivity, determined by the von Frey hair test, was observed during the PTU exposure and recovered after the exposure termination. The sciatic nerve compound action potential was also analyzed. Under single-pulse stimulation, no significant change in the threshold and conduction velocity was observed in the PTU-administered group. On the other hand, under train-pulse stimulation, the latency delay in the Aδ-fiber component was less in the PTU-administered group in Week 4 of PTU exposure, indicating relative hyperexcitability. Fluticasone, which is the anti-inflammatory agent with an ability to activate the voltage-gated potassium channel subfamily A (Kv1), restored the decrease in the latency change ratio by PTU exposure under the train-pulse stimulation supporting our hypothesis that Kv1 may be involved in the conductivity change. Kv1.1 protein level decreased significantly in the sciatic nerve of the PTU-administered group. These results indicate that adult-onset hypothyroidism causes mechanical hypersensitivity owing to hyperexcitability of the peripheral nerve and that reduction of Kv1.1 level may be involved in such alteration.

Dose-dependent immunomodulatory effects of bortezomib in experimental autoimmune neuritis.

Proteasome inhibition with bortezomib has been reported to exert an immunomodulatory action in chronic autoimmune neuropathies. However, bortezomib used for the treatment of multiple myeloma induces a painful toxic polyneuropathy at a higher concentration. Therefore, we addressed this controversial effect and evaluated the neurotoxic and immunomodulatory mode of action of bortezomib in experimental autoimmune neuritis. Bortezomib-induced neuropathy was investigated in Lewis rats using the von Frey hair test, electrophysiological, qPCR and histological analyses of the sciatic nerve as well as dorsal root ganglia outgrowth studies. The immunomodulatory potential of bortezomib was characterized in Lewis rats after experimental autoimmune neuritis induction with P253-78 peptide. Clinical, electrophysiological, histological evaluation, von Frey hair test, flow cytometric and mRNA analyses were used to unravel the underlying mechanisms. We defined the toxic concentration of 0.2 mg/kg bortezomib applied intraperitoneally at Days 0, 4, 8 and 12. This dosage induces a painful toxic neuropathy but preserves axonal regeneration in vitro. Bortezomib at a concentration of 0.05 mg/kg significantly ameliorated experimental autoimmune neuritis symptoms, improved experimental autoimmune neuritis-induced hyperalgesia and nerve conduction studies, and reduced immune cell infiltration. Furthermore, proteasome inhibition induced a transcriptional downregulation of Nfkb in the sciatic nerve, while its inhibitor Ikba (also known as Nfkbia) was upregulated. Histological analyses of bone marrow tissue revealed a compensatory increase of CD138+ plasma cells. Our data suggest that low dose bortezomib (0.05 mg/kg intraperitoneally) has an immunomodulatory effect in the context of experimental autoimmune neuritis through proteasome inhibition and downregulation of nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFKB). Higher bortezomib concentrations (0.2 mg/kg intraperitoneally) induce sensory neuropathy; however, the regeneration potential remains unaffected. Our data empathizes that bortezomib may serve as an attractive treatment option for inflammatory neuropathies in lower concentrations.

Beneficial effects of octreotide in alcohol-induced neuropathic pain. Role of H 2S, BDNF, TNF-α and Nrf2.

ABSTRACTPurposeTo explore the role and molecular mechanisms of neuroprotective effects of octreotide in alcohol-induced neuropathic pain.MethodsMale Wistar rats were employed and were administered a chronic ethanol diet containing 5% v/v alcohol for 28 days. The development of neuropathic pain was assessed using von Frey hair (mechanical allodynia), pinprick (mechanical hyperalgesia) and cold acetone drop tests (cold allodynia). The antinociceptive effects of octreotide (20 and 40 µg·kg–1) were assessed by its administration for 28 days in ethanol-treated rats. ANA-12 (0.25 and 0.50 mg·kg–1), brain-derived neurotrophic factor (BDNF) receptor blocker, was coadministered with octreotide. The sciatic nerve was isolated to assess the biochemical changes including hydrogen sulfide (H2S), cystathionine β synthase (CBS), cystathionine γ lyase (CSE), tumor necrosis factor-α (TNF-α), BDNF and nuclear factor erythroid 2-related factor 2 (Nrf2).ResultsOctreotide significantly attenuated chronic ethanol-induced neuropathic pain and it also restored the levels of H2S, CBS, CSE, BDNF, Nrf2 and decreased TNF-α levels. ANA-12 abolished the effects of octreotide on pain, TNF-α, BDNF, Nrf2 without any significant effects on H2S, CBS, CSE.ConclusionsOctreotide may attenuate the behavioral manifestations of alcoholic neuropathic pain, which may be due to an increase in H2S, CBS, CSE, BDNF, Nrf2 and a decrease in neuroinflammation.

The Involvement of Central Noradrenergic Pathway in the Analgesic Effect of Bee Venom Acupuncture on Vincristine-Induced Peripheral Neuropathy in Rats.

Vincristine is a vinca alkaloid anti-mitotic drug with a broad spectrum of effects on solid and hematologic cancers. The major dose-limiting factor of this anti-cancer regimen is painful peripheral neuropathy. However, no gold-standard analgesic option has been used clinically. In this study, we investigated the effects and mechanism of bee venom acupuncture (BVA) to alleviate peripheral neuropathic pain induced by repeated intraperitoneal infusions of vincristine (1 mg/kg/day, days 1–5 and 8–12) in rats. Subcutaneous injection with bee venom (BV, 1.0 mg/kg) at the ST36 acupoint ameliorated cold and mechanical hypersensitivity (i.e., aberrant withdrawal responses in acetone drop and von Frey hair tests, respectively). In vivo extracellular recording demonstrated that BVA inhibited cutaneous cold (acetone) and mechanical (brush, press, and pinch) stimuli-elicited abnormal hyperexcitation of the spinal wide dynamic range (WDR) neurons in vincristine-treated rats. In addition, the microinjection of lidocaine into the ipsilateral locus coeruleus or the antagonism of the spinal α2-adrenergic receptors clearly reversed the effects of BVA on cold and mechanical hypersensitivity, indicating a vital role of the descending noradrenergic modulation in analgesia. These findings suggest that BVA could be a potential therapeutic option for vincristine-induced peripheral neuropathy.

Locally Applied Stem Cell Exosome-Scaffold Attenuates Nerve Injury-Induced Pain in Rats.

PurposeNerve injury-induced pain is difficult to treat. In this study, we developed an alginate scaffold with human umbilical cord mesenchymal stem cell exosomes (EX-SC) to treat nerve injury-induced pain.Materials and MethodsThe scaffold was prepared and characterized for its physical traits and biocompatibility. In vitro studies of PC12 and HEK293 cells were used to evaluate the neuroprotective and neurotrophic effects of exosomes. Right L5/6 spinal nerve ligation (SNL) was performed in Sprague-Dawley rats to induce mechanical allodynia and thermal hyperalgesia, evaluated by von Frey hair and radiant heat tests. The EX-SC was wrapped around ligated L5/6 spinal nerves for treatment. Western blotting and immunofluorescence staining were used to evaluate neuron/glial activation, cytokines and neurotrophic factor of affected dorsal root ganglion (DRG).ResultsIn cell culture assay, the exosomes induce neurite outgrowth of PC12 cells and protect PC12 and HEK293 cells against formaldehyde acid treatment. On post-ligation day 21, rats receiving EX-SC had significantly higher median (interquartile range) withdrawal threshold and latency [14.1 (13.7–15.5) g, 14.2 (13.7–15.3) s] than saline-SC-treated rats [2.1 (1.7–3.0) g, 2.0 (1.8–2.4) s, P=0.02 and 0.002]. The EX-SC also attenuated SNL-induced up-regulation of c-Fos, GFAP, Iba1, TNF-α and IL-1β, while enhancing the level of IL-10 and GDNF, in the ipsilateral L5/6 DRG. After implantation for 21 days, the EX-SC enhanced the expression of myelin basic protein and IL-10 in injured L5/6 axons.ConclusionWe demonstrate the EX-SC possesses antinociceptive, anti-inflammation and pro-neurotrophic effects in the SNL pain model. It could be a promising therapeutic alternative for nerve injury-induced pain.

Long-Lasting and Additive Analgesic Effects of Combined Treatment of Bee Venom Acupuncture and Venlafaxine on Paclitaxel-Induced Allodynia in Mice.

Paclitaxel, a primary chemotherapeutic agent used to treat numerous solid malignancies, is commonly associated with debilitating peripheral neuropathy. However, a satisfactory gold-standard monotherapy for this neuropathic pain is not currently available. A combination strategy of two or more medications with different properties may achieve more beneficial effects than monotherapy. Thus, we investigated the analgesic efficacies and spinal mechanisms of the combination strategy, including bee venom acupuncture (BVA) and venlafaxine (VLX) against paclitaxel-induced allodynia in mice. Four intraperitoneal infusions of paclitaxel on alternating days (2 mg/kg/day) induced cold and mechanical allodynia for at least 1 week as assessed using acetone and the von Frey hair test, respectively. Co-treatment of BVA (1.0 mg/kg, s.c., ST36) with VLX (40 mg/kg, i.p.) at the medium dose produced a longer-lasting and additive effect than each monotherapy at the highest dose (BVA, 2.5 mg/kg; VLX, 60 mg/kg). Spinal pre-administration of idazoxan (α2-adrenergic receptor antagonist, 10 μg), methysergide (mixed 5-HT1/5-HT2 receptor antagonist, 10 μg), or MDL-72222 (5-HT3 receptor antagonist, 10 μg) abolished this analgesia. These results suggest that the combination therapy with BVA and VLX produces long-lasting and additive analgesic effects on paclitaxel-induced allodynia, via the spinal noradrenergic and serotonergic mechanism, providing a promising clinical strategy.

Spinal interleukin-6 contributes to central sensitisation and persistent pain hypersensitivity in a model of juvenile idiopathic arthritis

Pain is the most debilitating symptom in juvenile idiopathic arthritis. As pain correlates poorly to the extent of joint pathology, therapies that control joint inflammation are often inadequate as analgesics. We test the hypothesis that juvenile joint inflammation leads to sensitisation of nociceptive circuits in the central nervous system, which is maintained by cytokine expression in the spinal cord. Here, transient joint inflammation was induced in postnatal day (P)21 and P40 male Sprague-Dawley rats with a single intra-articular ankle injection of complete Freund’s adjuvant. Hindpaw mechanical pain sensitivity was assessed using von Frey hair and weight bearing tests. Spinal neuron activity was measured using in vivo extracellular recording and immunohistochemistry. Joint and spinal dorsal horn TNFα, IL1β and IL6 protein expression was quantified using western blotting. We observed greater mechanical hyperalgesia following joint inflammation in P21 compared to P40 rats, despite comparable duration of swelling and joint inflammatory cytokine levels. This is mirrored by spinal neuron hypersensitivity, which also outlasted the duration of active joint inflammation. The cytokine profile in the spinal cord differed at the two ages: prolonged upregulation of spinal IL6 was observed in P21, but not P40 rats. Finally, spinal application of anti-IL-6 antibody (30 ng) reduced the mechanical hyperalgesia and neuronal activation. Our results indicate that persistent upregulation of pro-inflammatory cytokines in the spinal dorsal horn is associated with neuronal sensitisation and mechanical hyperalgesia in juvenile rats, beyond the progress of joint pathology. In addition, we provide proof of concept that spinal IL6 is a key target for treating persistent pain in JIA.

Aquaporin 4 is involved in chronic pain but not acute pain

Accumulating evidence has revealed that spinal glia plays an important role in the processing of pain, particularly chronic pain. Aquaporin 4 (AQP4), the predominant water channel exists in astrocytes, has been proved to modulate astrocytic function and thus participate in many diseases of the central nervous system. However, there is still controversy over whether AQP4 is involved in pain modulation. In the present study, we investigated the effects of AQP4 on pain by examining chronic inflammatory pain, neuropathic pain, and thermal, chemical, and mechanical stimuli-induced acute pain in AQP4 knockout mice. In Complete Freund’s adjuvant-induced chronic inflammatory pain and spared nerve injury-induced neuropathic pain models, AQP4−/− mice attenuated pain-related behavioral responses compared with AQP4+/+ mice, demonstrating that AQP4 deficiency relieved chronic inflammatory pain and neuropathic pain. In the tail-flick and hot-plate tests, two acute pain models of thermal stimuli, no differences in pain-related behaviors were detected between AQP4+/+ and AQP4−/− mice. In the formalin and capsaicin tests, two models of chemical stimuli-induced acute pain, no differences in the durations of licking the injected hindpaw were found between AQP4+/+ and AQP4−/− mice. In the von Frey hair test, a model of mechanical stimuli-induced acute pain, no significant differences in withdrawal thresholds were found between these two genotypes mice as well. These results indicated that AQP4 deficiency did not affect acute pain induced by thermal, chemical, and mechanical stimuli. Taken together, our findings suggested that AQP4 contributes to chronic pain, but not acute pain.

Intermediate Cervical Plexus Block in the Management of Persistent Postoperative Pain Post Carotid Endarterectomy: A Prospective, Randomized, Controlled, Clinical Trial

Background: The mechanisms of persistent postoperative pain (PPP) with neuropathic features after carotid endarterectomy (CEA) are multifaceted and are incompletely understood. Objectives: The aim of this research was to assess whether the ultrasound-guided (USG) intermediate cervical plexus block (ICPB) could provide better control of PPP and neuropathic disturbances (NPDs) after CEA than the USG superficial cervical plexus block (SCPB). Study Design: Prospective, randomized, controlled, clinical trial. Setting: This clinical trial was conducted at the SS Filippo and Nicola Academic Hospital of Avezzano (L’Aquila, Italy). Methods: Patients who were scheduled for primary CEA were chosen. In the experimental group, the USG-ICPB was performed unilaterally, at the level of the third cervical vertebra. The needle was inserted into the deep lamina of the deep fascia of the neck, between the posterior border of the middle scalene muscle and the anterior border of the posterior scalene muscle. Three milliliters saline solution was injected into the opening of the deep lamina, and 20 mL 0.375% levobupivacaine was injected. In the control group, the anesthetic target was located at the inferior border of the sternocleidomastoid muscle at the level of the third cervical vertebra. The needle was superficially inserted below the skin, and 2 to 3 mL saline solution was injected into the opening of the superficial lamina of the deep fascia of the neck. A total of 20 mL 0.375% isobaric levobupivacaine was subsequently injected. The primary outcome measure was the proportion of patients with PPP on movement and at rest 3 months after surgery. The secondary outcome measures were NPD assessment scores using the von Frey hair test and the Lindblom test, opioid and pregabalin consumption. Adverse effects were also recorded. Results: A total of 98 consecutive patients were enrolled and randomized to receive either a USGSCPB (control group, n = 49) or a USG-ICPB (experimental group, n = 49). The sensory blockade was longer in the experimental group. Three months after surgery, the proportions of patients with PPP on movement were significantly different between the experimental and control groups (33%, 95% confidence intervals [CI], 20%-47% vs. 71%, 95% CI, 57%-83%; P < 0.001), whereas there were no differences in the proportions of patients with pain at rest between groups (31%, 95% CI, 18%-45% vs. 49%, 95% CI, 34%-64%; P = 0.063). The proportions of patients with NPDs were not different between the groups, whereas the sizes of the areas of interest (cm2 ) were significantly different. Limitations: A limitation of this study is that we assessed NPDs for only 3 months using the von Frey hair test and the Lindblom test without additional instrumental techniques. Additionally, there are many risk factors for NPDs after CEA. For this reason, another limitation of this research is that we neglected to consider the relationship between the choice of anesthetic block and the presence of these risk factors.Conclusions: The USG-ICPB provided long-lasting analgesia during the postoperative period and might mitigate the development of NPDs, thereby decreasing the analgesic drug requirement. Key words: Carotid endarterectomy, intermediate cervical plexus block, myofascial planes of neck, neuropathic disturbances, persistent postoperative pain, superficial cervical plexus blocks, ultrasound guidance, vascular disease

CircStrn3 is involved in bone cancer pain regulation in a rat model.

Bone cancer pain (BCP) is a common chronic pain that is caused by a primary or metastatic bone tumor. More detailed molecular mechanisms of BCP are warranted. In this study, we established a BCP rat model. The von Frey hair test, body weight, and hematoxylin and eosin staining were employed. We screened differentially expressed circRNAs (DECs) between the BCP group and sham group. The results revealed that 850 DECs were significantly up-regulated and 644 DECs were significantly down-regulated in the BCP group. Furthermore, we identified 1177 differentially expressed genes (DEGs) significantly up-regulated and 565 DEGs significantly down-regulated in the BCP group. Gene Ontology annotation of all 1742 DEGs revealed that biological regulation of metabolic processes, cellular processes, and binding were the top enriched terms. For Kyoto Encyclopedia of Genes and Genomes analysis, phagosome, HTLV-I infection, proteoglycans in cancer, and herpes simplex infection were significantly enriched in this study. In addition, we identified four selected circRNAs, chr6:72418120|72430205, chr20:7561057|7573740, chr18:69943105|69944476, and chr5:167516581|167558250, by quantitative real time PCR. chr6:72418120|72430205 (circStrn3) was selected for further study based on expression level and the circRNA–miRNA–mRNA network table. Western blot analysis suggested that knockdown of circStrn3 could effectively induce Walker 256 cell apoptosis. In summary, our study provided a more in-depth understanding of the molecular mechanisms of BCP.

Effect of m-trifluoromethyl-diphenyl diselenide on acute and subchronic animal models of inflammatory pain: Behavioral, biochemical and molecular insights

m-Trifluoromethyl-diphenyl diselenide [(m-CF3-PhSe)2] is an organoselenium molecule that displays multiple pharmacological actions, including the antinociceptive effect. The current study investigated the (m-CF3-PhSe)2 restorative properties in models of acute and chronic inflammatory pain induced by complete Freund's adjuvant (CFA). Male adult Swiss mice received an intraplantar injection of CFA in the hindpaw and 24 h (acute) or 14 days (subchronic) later they were treated with a single or repeated (m-CF3-PhSe)2 schedule via intragastric route, respectively. The mechanical and thermal hypernociceptive behaviors were assessed by von Frey hair and hot plate tests. Samples of injected paw were collected to evaluate the tissue edema and myeloperoxidase (MPO) activity while cerebral contralateral cortex samples were used to determine the inflammatory proteins content (subchronic protocol). The acute (m-CF3-PhSe)2 administration (1 and 10 mg/kg) reduced the hypernociceptive behavior and both paw thickness and MPO activity induced by CFA injection. In the subchronic protocol, the repeated administration with a low effective dosage of (m-CF3-PhSe)2 reduced the mechanical and thermal hypernociception as well as restored the edema and MPO activity in paw samples. In addition, the repeated treatment schedule mitigated the increase in TNF-α, IL-1β and COX-2 content in cerebral contralateral cortex induced by CFA injection. Collectively, these data showed that (m-CF3-PhSe)2 presents anti-inflammatory properties, which could be mediated by an interplay between peripheral and central mechanisms of action, reinforcing the potential biological properties of the compound.

GPR34 in spinal microglia exacerbates neuropathic pain in mice

BackgroundNeuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain.MethodsmRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography–tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity.ResultsGPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain.ConclusionsInhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

The Analgesic Effect of Venlafaxine and Its Mechanism on Oxaliplatin-Induced Neuropathic Pain in Mice.

The analgesic effect of venlafaxine (VLX), which is a selective serotonin and noradrenaline reuptake inhibitor (SNRI), has been observed on oxaliplatin-induced neuropathic pain in mice. Significant allodynia was shown after oxaliplatin treatment (6 mg/kg, i.p.); acetone and von Frey hair tests were used to assess cold and mechanical allodynia, respectively. Intraperitoneal administration of VLX at 40 and 60 mg/kg, but not 10 mg/kg, significantly alleviated these allodynia. Noradrenaline depletion by pretreatment of N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 50 mg/kg, i.p.) blocked the relieving effect of VLX (40 mg/kg, i.p.) on cold and mechanical allodynia. However, serotonin depletion by three consecutive pretreatments of para-chlorophenylalanine (PCPA, 150 mg/kg/day, i.p.) only blocked the effect of VLX on mechanical allodynia. In cold allodynia, the α2-adrenergic antagonist idazoxan (10 μg, i.t.), but not the α1-adrenergic antagonist prazosin (10 μg, i.t.), abolished VLX-induced analgesia. Furthermore, idazoxan and 5-HT3 receptor antagonist bemesetron (MDL-72222, 15 μg, i.t.), but not prazosin or mixed 5-HT1, 2 receptor antagonist methysergide (10 μg, i.t.), abolished VLX-induced analgesia in mechanical allodynia. In conclusion, 40 mg/kg of VLX treatment has a potent relieving effect against oxaliplatin-induced neuropathic pain, and α2-adrenergic receptor, and both α2-adrenergic and 5-HT3 receptors are involved in this effect of VLX on cold and mechanical allodynia, respectively.